A lysosome-Targeting dual-functional fluorescent probe for imaging intracellular viscosity and beta-Amyloid

Article abstract of DOI:10.1039/c9cc00113a

A lysosome-Targeting dual-functional fluorescent probe was rationally designed and developed for imaging intracellular lysosomal viscosity and beta-Amyloid. More importantly, the real-Time tracking of the dynamic movement of lysosomes, as vesicle structures, has been achieved using Lyso-MC.

Full text of DOI:10.1039/c9cc00113a

View Article Online
View Journal
ChemComm
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: H. Tan, Y. Qiu, H.
Sun, J. Yan and L. Zhang, Chem. Commun., 2019, DOI: 10.1039/C9CC00113A.
This is an Accepted Manuscript, which has been through the
Volume 52 Number
1 4 January 2016 Pages 1–216
Royal Society of Chemistry peer review process and has been
accepted for publication.
ChemComm
Accepted Manuscripts are published online shortly after
Chemical Communications
www.rsc.org/chemcomm
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
ISSN 1359-7345
COMMUNICATION
Marilyn M. Olmstead, Alan L. Balch, Josep M. Poblet, Luis Echegoyenet al.
Reactivity differences of Sc3N@C2n (2n 68 and 80). Synthesis of the
first methanofullerene derivatives of Sc N@D5h-C80
=
3
rsc.li/chemcomm

Please do not adjust margins
Page 1 of 4
ChemComm
View Article Online
DOI: 10.1039/C9CC00113A
Journal Name
COMMUNICATION
A lysosome-targetable dual-functional fluorescent probe for
imaging intracellular viscosity and beta-amyloid
Hui-ya Tan,a Yu-tai Qiu,a Han Sun, a Jin-wu Yan, *a,b,c and Lei Zhang *a,b,c
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
A lysosome-targetable dual-functional fluorescent probe was accumulation in pathological conditions.11 Besides, much
rationally designed and developed for imaging intracellular evidence advocates that the lysosomal system, an acidic
lysosomal viscosity and beta-amyloid. More importantly, the real- vesicular compartment containing various hydrolases, is related
time tracking of dynamic movement of lysosome, as vesicle to Aβ production and neurotoxicity.16,17 In addition, restoring
structure, has been achieved by using Lyso-MC.
autophagy or improving lysosomal degradation of intracellular
Aβ may be a potential therapeutic approach to treat AD.10,13
Therefore, monitoring cellular lysosomal movement or
physiological changes and intracellular Aβ could provide a
better understanding for AD pathology and its early diagnosis.
It is reported that the lysosomal viscosity is an important
benchmark for lysosome situation and it reflects the status and
function of this organelle,16,18,19 therefore, tracing lysosome or
monitoring its viscosity changes is of great benefit for AD
diagnosis and pathological studies. Recently, many Aβ
fluorescent probes20-22 and many lysosome-targetable
fluorescent probes imaging viscosity19,23,24 have been reported.
However, as we know, the lysosome-targetable dual-functional
fluorescent probe imaging both viscosity and Aβ aggregates at
cellular level has not been reported.
Alzheimer’s disease (AD), an age-related pathology, leads to
progressive cognitive decline, communication barrier, language
disorder and loss of self-care ability.1-3 Besides, AD has affected
millions of people worldwide with 5 million new cases every
year,4 however, the symptomatic treatment only mildly
improves the symptoms of AD but still incurable due to the
unclear pathophysiological mechanism of AD.5,6 The toxic
amyloid-β peptide (Aβ) generates from APP (Amyloid Precursor
Protein) after the sequential action of β- and γ-secretase and
accumulates constantly to the senile plaques, the hallmark of
AD, which are considered to be basic pathological targets for
primary prevention, early diagnosis and treatment of the
disease.5,6 It has been pointed out that intracellular Aβ is
involved in the early stage of AD and spreads from neuron to
neuron, directly causing neurotoxicity and triggering AD
pathology.7-9 Several studies have found that amyloid precursor
protein (APP) and amyloid-β peptide (Aβ) are generated from
autophagy and endocytic pathway,10-13 besides, AD is
characterized by lysosomal dysfunction and a massive
accumulation of lysosomal-related vesicles in the degenerating
neurons.11,14,15 Moreover, increasing studies point out that the
dysfunction of autophagy pathway would be conducive to Aβ
Herein, a fluorescent probe (Lyso-MC) for sensitively imaging
intracellular lysosome and Aβ aggregates was reasonably
25
designed and developed. The neutral merocyanine dye (MC-1
)
was selected as the fluorophore to design this probe, which was
reported previously by our group as a good scaffold imaging Aβ
plaques in vivo and imaging viscosity at cellular level reported
by Lin’s group.26 However, the low quantum yield and photo-
stability of MC-1 restricted its further application. In this study,
3-morpholinopropylamine moiety was rationally introduced as
electron-donor to enhance the conjugation of π electron so as
a. School of Biology and Biological Engineering, South China University of
Technology, Guangzhou 510006, P.R. China.
Guangdong Provincial Engineering and Technology Research Center of
O
N
b.
Cl
Biopharmaceuticals, South China University of Technology, Guangzhou 510006,
P.R. China.
CN
CN
NH
N
CN
CN
c. Joint International Research Laboratory of Synthetic Biology and Medicine, South
China University of Technology, Guangzhou 510006, P.R. China. E-mail:
yjw@scut.edu.cn; lzhangce@scut.edu.cn.
N
Electronic Supplementary Information (ESI) available: Synthesis and
characterization of probe, experimental procedures, and supplemental spectra and
graphs. See DOI: 10.1039/x0xx00000x
MC-1
Lyso-MC
Scheme 1 Chemical structures of MC-1 and Lyso-MC.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
ChemComm
Page 2 of 4
COMMUNICATION
Journal Name
S5(ESI†), less fluorescence responses were obVsieewrvAertdicleuOpnloinne
DOI: 10.1039/C9CC00113A
interaction with BSA, HSA and Aβ monomer, indicating low
nonspecific binding and its good selectivity towards Aβ1-42
aggregates. Next, the interaction between Lyso-MC and Aβ1-42
aggregates were further studied by the fluorescence titrations.
As shown in the Fig. 1C, upon titration with Aβ1-42 aggregates,
the emission wavelength blue-shifted to 610 nm and the
emission intensity was significantly enhanced. Besides, it
showed a good linear relationship (R2 = 0.9899) with Aβ1-42
aggregates concentration ranging from 0 to 2.5 μM (Fig. 1D).
Moreover, the detection limit of Lyso-MC was determined to be
as low as 10.2 nM (Table S2, ESI†). After the saturation binding
assay between Lyso-MC and Aβ1-42 aggregates, the binding
constant was calculated to be 31.74 nM, indicating that the
probe Lyso-MC exhibited moderate affinity towards Aβ
aggregates (Table S2, ESI†). In addition, the LogP value was
measured to be 2.63, predicting that Lyso-MC had its potential
in good blood brain barrier (BBB) penetration. Besides, the red
and blue shift was observed for Lyso-MC in viscous and
hydrophobic environments respectively, therefore it could be
Fig. 1 (A) Solvent viscosity-dependent fluorescence changes of Lyso-MC (0.25 μM) in
water-glycerol system; (B) The linear relationship between log I620 and log η of Lyso-
MC; (C) Fluorescence spectroscopic titration of Lyso-MC (0.25 μM) towards Aβ1-42
aggregates in PBS buffer (pH=7.4) ; (D) Emission intensity of Lyso-MC as a function of
the concentration of Aβ1-42 aggregates in PBS buffer (pH=7.4).
to improve the fluorescence quantum yield, and functioned as
lysosomal targeting group19,23,24. As we know, Lyso-MC was the
first lysosome-targetable dual-functional probe monitoring
viscosity and Aβ aggregates at cellular level.
The synthesis of Lyso-MC is outlined in Scheme S1 (ESI†) and
1
its structure (Scheme 1) was confirmed by H and 13C NMR
spectroscopy and HR-MS. The fluorescence response spectra of
Lyso-MC were first investigated in a water-glycerol system with
increasing viscosity. As shown in Fig. 1A, Lyso-MC emitted at
615 nm along with the weakest fluorescence in an absolute
water medium. Meanwhile, the emission intensity showed a
dramatic fluorescence enhancement (33-fold, Table S1, ESI†)
and the emission wavelength red-shifted to 620 nm due to the
viscous increasement which was caused by the increasing ratio
of glycerol in water-glycerol system. In addition, as shown in Fig.
1B, the good linear relationship between log I620 and log η (R2 =
0.99, x = 0.53) was quantitatively gained according to the
Förster-Hoffmann equation. Moreover, as shown in the Fig. S2
(ESI†), the polarity environment only had an influence on the
emission wavelength where Lyso-MC showed higher absorption
and emission wavelengths when increasing the polarity of the
surrounding environment. Compared with the strong
fluorescence of Lyso-MC in glycerol, its fluorescence in other
solvents with different polarities was negligible (Fig. S3, ESI†),
therefore, its fluorescence intensity was insensitive to polarity
changes. In the pH titration experiment, Lyso-MC could not be
disturbed by the lysosomal pH range (pH = 4.5~5.0) and only
affected by viscosity (Fig. S4, ESI†). Besides, Lyso-MC was not
interfered by various amino acids and ions (Fig. S5, ESI†),
exhibiting good selectivity towards viscosity. These results
indicated that the Lyso-MC was a stable viscosity fluorescent
probe for detecting viscosity in lysosomes.
Fig. 2 (A) Colocalization fluorescence images of SH-SY5Y cells co-incubated with Lyso-
MC (5 μM) and Lyso Tracker Green (1 μM); (B) Colocalization fluorescence images of
SH-SY5Y cells co-incubated with Lyso-MC (5 μM) and Mito Tracker Green (1 μM).
Scale bars: 20 μm; (C) Confocal fluorescence images of SH-SY5Y cells incubated with
o
Lyso-MC (5 μM) only at 37 C; (D) Confocal fluorescence images of SH-SY5Y cells
incubated with Lyso-MC (5 μM) only at 4 o C; (E) Cells were incubated with Lyso-MC
(5 μM) only at 37 o C for 30 min following by treatment with dexamethasone (5 μM)
for another 10 min; (F) Cells were incubated with Lyso-MC (5 μM) only at 37 o C for
30 min following by treatment with DMSO (10 μL) for another 30 min; (G) Analysis of
the relative fluorescence intensity in SH-SY5Y cells treated in different incubation
situations (n=3, data were obtained from ImageJ Software), Scale bars: 5 μm.
We further investigated the fluorescence response of Lyso-
MC towards the synthetic Aβ1-42 aggregates, Aβ monomer, BSA
and HSA. As expected, the probe exhibited a remarkable (10-
fold) fluorescence intensity in the presence of Aβ1-42 aggregates
at a low final concentration of 0.25 μM. As displayed in Fig.
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 3 of 4
ChemComm
Please do not adjust margins
Journal Name
COMMUNICATION
used as dual-functional fluorescent probe to distinguish as a clinic drug has been proved to be effective ViinewiAnrcticrleeaOsnilninge
viscosity changes and Aβaggregates on the basis of the different lysosomal viscosity.27,28 With the aim toDinOvI:e1s0t.i1g0a3t9e/Ct9hCeC0a0b1il1i3tAy
emission wavelengths in the different environments.
of Lyso-MC to monitor the variation of intracellular lysosomal
Due to its good response to viscosity, we further investigated viscosity, the living SH-SY5Y cells was first incubated with the
the application of Lyso-MC in living cells. Before fluorescent probe (5 μM) at different temperatures. As shown in Fig. 2, the
o
imaging in living cells, the cytotoxicity of Lsyo-MC was first cells incubated at 4 C exhibited stronger red fluorescence in
assessed in SH-SY5Y and Hela cells. A standard MTT assay in SH- red channel when comparing with cells at 37 o C due to the
SY5Y and Hela cells showed that over 80% cells remained alive greater viscosity value at 4 o C. As a result, Lyso-MC could detect
when 12.5 μM Lyso-MC was internalized for 12 h (Fig. S7, ESI†), the lysosomal viscosity in living cells. Besides, it was clearly
indicating that the probe exhibited weak cytotoxicity. Then, the found that the fluorescence intensity increased after treatment
fluorescence imaging of Lyso-MC in living SH-SY5Y and Hela cells with dexamethasone (5 μM), indicating that the lysosomal
was performed by confocal laser scanning microscopy. The cells viscosity value was larger than that at 37 o C. In addition, there
exhibited negligible fluorescence in the absence of Lyso-MC
,
were no obvious fluorescence changes when incubated with
o
while the cells incubated with Lyso-MC exhibited strong red DMSO (10 μL) compared with incubated with probe at 37 C,
fluorescence and the fluorescence intensity was dose and time which further demonstrated that the probe could monitor the
dependent, indicating that Lyso-MC permeated the membrane variation of lysosomal viscosity without the interference of
and can stain living cells (Fig. S8 and Fig. S9, ESI†).
polarity fluctuation in living cells.
Encouraged by the good membrane-permeable of the probe,
Due to good lysosome-targetable performance of the probe,
we further investigated the co-localization of Lyso-MC in we investigated the ability of the probe to trace the lysosomes
lysosomes of SH-SY5Y cells with commercially available Lyso- in real time and observed the subcellular structure of lysosome
tracker Green DND-26 and Mito-tracker Green FM. As shown in using CLSM with ultra-high-resolution module. The pictures of
Fig. 2, Lyso-MC overlapped very well with Lyso-Tracker Green SH-SY5Y cells incubated with Lyso-MC were taken every 4
with the overlap coefficient as high as 0.96 between Lyso-MC seconds by CLSM. As shown in Fig. 3, it demonstrated that
and Lyso-Tracker Green, while the coefficient between Lyso-MC dynamic lysosomes in living cells were vesicular structure and
and Mito-Tracker green was only 0.53. The results indicated five different pseudo-colors were intended to clearly show the
that Lyso-MC was a lysosome-targetable probe. Meanwhile, we movements of lysosome at various time points (0, 20, 40, 60 and
investigated the photo-stability of Lyso-MC inside cells, which 80 s). Merged images at various time points and dynamic video
was of great importance for imaging the viscosity of lysosome. indicated that the movements of lysosome were rather active
Then the commercially available Lyso-Tracker Green was used and Lyso-MC could trace intracellular lysosome in real-time.
as a comparison. The SH-SY5Y cells were exposed to 561 nm
Considering the specific binding of the probe to Aβ1-42
channel after incubating with Lyso-MC while the Lyso-Tracker aggregates in solution and important pathological action of
Green group was exposed to 488 nm channel. After 10 min intracellular Aβ aggregates, the PC12 cells transfected for the
continuous illumination, the signal loss for Lyso-MC and Lyso- overexpression of Aβ (PC12-EGFP-Aβ cells) and the normal PC12
Tracker Green was 72% and 88%, respectively (Fig. S10, ESI†). cells taken as controls were conducted to investigate whether
Therefore, the probe exhibited moderate photo-stability both Lyso-MC could detect Aβ aggregates at cellular level. As shown
in solution (Fig. S6, ESI†) and in living cells.
in Fig. 11SB (ESI†), granular aggregates were detected with
It is known that a lower temperature contributes to the Lyso-MC in the cytoplasm, which was consistent with
intracellular viscosity increasement and dexamethasone used immunofluorescence group (Fig. 11SA, ESI†). Meanwhile, there
Fig. 3 (A-E) Confocal images of SH-SY5Y cells stained with 5 μM for 30 min. Different pseudo colors were used to illustrate the fluorescence images at 20 s, 40 s, 60 s and 80
s, respectively. (F-J) Merged of images at two different time points: (F) 0 and 20 s, (G) 20 and 40 s, (H) 40 and 60 s, (I) 60 and 80 s, (J) 0 and 80 s. Inset is the zoomed-in
image. Dark pink and light pink arrow heads indicate different size of lysosome. White arrow heads indicate the significant movements of lysosome. Scale bars: 25 μm.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
ChemComm
Page 4 of 4
COMMUNICATION
Journal Name
View Article Online
DOI: 10.1039/C9CC00113A
Conflicts of interest
There are no conflicts to declare.
Notes and references
1
X. Zhang, Y. Tian, C. Zhang, X. Tian, A. W. Ross, R. D. Moir, H.
Sun, R. E. Tanzi, A. Moore and C. Ran, Proc. Natl. Acad. Sci. U.
S. A., 2015, 112, 9734-9739.
2
3
4
5
6
7
8
C. Ballard, S. Gauthier, A. Corbett, C. Brayne, D. Aarsland and
E. Jones, The Lancet, 2011, 377, 1019-1031.
M. Staderini, M. A. Martin, M. L. Bolognesi and J. C. Menendez,
Chem. Soc. Rev., 2015, 44, 1807-1819.
H. Tong, K. Lou and W. Wang, Acta Pharm. Sin. B, 2015, 5, 25-
33.
Fig. 4 Co-localization images in transfected PC12 cells after immunofluorescence
for Aβ1-42 aggregates and then co-stained with Lyso-MC (1 μM). (A)
Immunofluorescence image from Alexa Fluor®405; (B) Fluorescence image from
EGFP; (C) Fluorescence image from Lyso-MC; (D) The merged image of (A), (B)
and (C); (E) The merged image of (A) and (C); (F) Intensity correlation plot of co-
stain Lyso-MC and immunofluorescence; ICA plots of (G) stain Lyso-MC and (H)
stain Alexa Fluor®405 of immunofluorescence. (Analyzed by Image J software)
F. Mangialasche, A. Solomon, B. Winblad, P. Mecocci and M.
Kivipelto, Lancet Neurol., 2010, 9, 702-716.
S. Gauthier, L. Wu, P. Rosa-Neto and J. Jia, Transl.
Neurodegener., 2012, 1, 1-13.
J. Naslund, V. Haroutunian, R. Mohs, K. L. Davis, P. Davies, P.
Greengard and J. D. Buxbaum, JAMA, 2000, 283, 1571-1577.
M. Koistinaho, M. Ort, J. M. Cimadevilla, R. Vondrous, B.
Cordell, J. Koistinaho, J. Bures and L. S. Higgins, Proc. Natl.
Acad. Sci. U. S. A., 2001, 98, 14675-14680.
was negligible fluorescence found in the control PC12 cell (Fig.
S12, ESI†). Besides, there was also no fluorescence observed in
the PC12-EGFP-Aβ cells with no staining with Lyso-MC or
immunofluorescence (Fig. S13, ESI†). We also conducted the co-
localization experiment for Aβ aggregates in the transfected
PC12-EGFP-Aβ cells through co-staining Lyso-MC after
immunofluorescence assay with secondary antibody Alexa
Fluor®405. As shown in Fig. 4, the Lyso-MC and Alexa Fluor®405
merged well with a good Pearson’s coefficient of 0.7997. The
ICA (intensity correlation analysis, which was used to evaluate
the intensity distribution of the two co-existing stains) plots for
the Lyso-MC and Alexa Fluor®405 created an unsymmetrical
hourglass-shaped scatterplot which tended to toward positive
values (Fig. 4G,H). No matter the ex situ monitor or co-localized
for Aβ aggregates, the probe Lyso-MC was confirmed to detect
Aβ aggregates at cellular level.
9
S. Nath, L. Agholme, F. R. Kurudenkandy, B. Granseth, J.
Marcusson and M. Hallbeck, J. Neurosci., 2012, 32, 8767-8777.
10 R. A. Nixon and A. M. Cataldo, J Alzheimers Dis., 2006, 9, 277-
289.
11 R. A. Nixon, J Cell Sci., 2007, 120, 4081-4091.
12 R. A. Nixon, D.-S. Yang and J.-H. Lee, Autophagy, 2008, 4, 590-
599.
13 F. Pickford, E. Masliah, M. Britschgi, K. Lucin, R. Narasimhan,
P. A. Jaeger, S. Small, B. Spencer, E. Rockenstein, B. Levine and
T. Wyss-Coray, J. Clin. Invest., 2008, 118, 2190-2199.
14 K. Inoue, J. Rispoli, H. Kaphzan, E. Klann, E. I. Chen, J. Kim, M.
Komatsu and A. Abeliovich, Mol. Neurodegener., 2012, 7, 48.
15 J.-H. Lee, W. H. Yu, A. Kumar, S. Lee, P. S. Mohan, C. M.
Peterhoff, D. M. Wolfe, M. Martinez-Vicente, A. C. Massey, G.
Sovak, Y. Uchiyama, D. Westaway, A. M. Cuervo and R. A.
Nixon, Cell, 2010, 141, 1146-1158.
16 R. A. Nixon, A. M. Cataldo and P. M. Mathews, Neurochem.
Res., 2000, 25, 1161-1172.
17 J. Han and K. Burgess, Chem. Rev., 2010, 110, 2709-2728.
18 T. Liu, X. Liu, D. R. Spring, X. Qian, J. Cui and Z. Xu, Sci. Rep.,
2014, 4, 5418.
19 L.-L. Li, K. Li, M.-Y. Li, L. Shi, Y.-H. Liu, H. Zhang, S.-L. Pan, N.
Wang, Q. Zhou and X.-Q. Yu, Anal. Chem., 2018, 90, 5873-5878.
20 J.-y. Zhu, L.-f. Zhou, Y.-k. Li, S.-b. Chen, J.-w. Yan and L. Zhang,
Anal. Chim. Acta, 2017, 961, 112-118.
21 K. Rajasekhar, N. Narayanaswamy, N. A. Murugan, K. Viccaro,
H.-G. Lee, K. Shah and T. Govindaraju, Biosens. Bioelectron.,
2017, 98, 54-61.
22 Y. Li, K. Wang, K. Zhou, W. Guo, B. Dai, Y. Liang, J. Dai and M.
Cui, Chem. Commun., 2018, 54, 8717-8720.
23 B. Guo, J. Jing, L. Nie, F. Xin, C. Gao, W. Yang and X. Zhang, J.
Mater. Chem. B, 2018, 6, 580-585.
24 L. Hou, P. Ning, Y. Feng, Y. Ding, L. Bai, L. Li, H. Yu and X. Meng,
Anal. Chem., 2018, 90, 7122-7126.
25 J.-w. Yan, J.-y. Zhu, K.-x. Zhou, J.-s. Wang, H.-y. Tan, Z.-y. Xu,
S.-b. Chen, Y.-t. Lu, M.-c. Cui and L. Zhang, Chem. Commun.,
2017, 53, 9910-9913.
In conclusion, a red-emitting dual-functional fluorescent
probe with low cytotoxicity based on a neutral merocyanine dye
(
MC-1) was rationally designed and developed for imaging
intracellular lysosomal viscosity and Aβ aggregates. The spectral
results showed that Lyso-MC exhibited an excellent linear
relationship between the logarithm of fluorescence intensity
and the logarithm of viscosity (R2 = 0.99, x=0.53), meanwhile, it
showed significant turn-on fluorescence response when bound
to Aβ aggregates and high sensitivity towards Aβ aggregates
(detection limit, 10.2 nM). Besides, Lyso-MC has been applied
to monitor lysosomal viscosity changes and detect intracellular
Aβ aggregates. More importantly, the real-time tracking of
dynamic movement of lysosome, as vesicle structure, has been
achieved by using Lyso-MC. Taking advantage of its remarkable
properties, the versatile probe could be a promising imaging
tool in the Alzheimer’s disease pathology related field.
This work was financially supported by the Natural Science
Foundation of Guangdong Province, China (2016A030310463
and 2016A010121004), the National Natural Science
Foundation of China (21502056) and the Medical Scientific
Research Foundation of Guangdong Province, China (A2018080)
26 R. Guo, J. Yin, Y. Ma, G. Li, Q. Wang and W. Lin, Sen. and
Actuators B: Chem.l, 2018, 271, 321-328.
27 S. Humphries, Proc. Natl. Acad. Sci. U. S. A., 2013, 110, 14693-
14698.
。
28 L. Wang, Y. Xiao, W. Tian and L. Deng, J. Am. Chem. Soc., 2013,
135, 2903-2906.
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins